Summary The objective of this study was to adapt an assay to measure L‐gulonolactone oxidase (GLO) activity, the final step in ascorbate synthesis, in chickens and use the procedure to study the effect of gender, age, and food deprivation. To adapt a procedure used for mammalian tissues the kinetic constants were estimated using chicken kidney and assay conditions optimized in terms of storage of tissue and homogeneity of distribution. A series of experiments were conducted using the standardized assay with individual animals as the experimental unit. Gender differences were detected only in mature birds with male chickens having lower GLO activity than females. Time‐course changes in GLO activity in immature chicks were best characterized by a segmented response function consisting of a parabola joined to a straight line. The maximum value was estimated to occur at 13 days and from day 16 declined linearly. The functional relationship did not provide evidence of an early lag period or a peak at 4 weeks in immature chickens. Food deprivation in matched groups of birds demonstrated the effect of food on GLO activity in chickens. Food and water deprivation for 12 h and food deprivation for 24 h had no significant effect on GLO activity. Food deprivation for 48 h caused a significant decrease in GLO activity and it was not further depressed by deprivation for 72 h. Initial GLO activity was restored within 24 h of repletion. The results clearly demonstrated that gender, age, and starvation are determinants of ascorbate synthesis in chickens.